Several paths of evidence converge in implicating a role for the cholinergic system in the pathophysiology of affective illness. In both unipolar depressed and euthymic bipolar subjects, cholinomimetic drugs (i.e., muscarinic agonists, acetylcholinesterase inhibitors) exacerbate depressive signs and symptoms such as dysphoria, psychomotor retardation, impairment of attention and memory, hypothalamic pituitary adrenal axis hyperactivity and sleep EEG abnormalities (Janowsky et al., 1974;Overstreet, 1993). In healthy subjects, the acetylcholinesterase inhibitor physostigmine elicits a range of depressive symptoms including dysphoria, anergia, psychomotor slowing, emotional lability, sleep disturbances, memory and concentration impairment, and with higher doses, tearfulness and depression. These effects have been shown to reflect stimulation of muscarinic receptors (Davis et al., 1976;Oppenheim et al., 1979;Risch et al., 1981a). Cholinomimetics also exacerbate behavioral despair in putative animal models of depression. Conversely, the anticholinergic agent biperidine improved symptoms of depression in a placebo controlled study (Fleischhacker et al., 1987). Moreover, muscarinic cholinomimetics and a choline rich nutrient, lecithin (phosphatidylcholine) exert antimanic effects in bipolar subjects. Potentially consistent with these observations, depressed subjects exhibit hypersensitivity to cholinomimetic agents. Administration of muscarinic cholinergic agonists, ACh releasing agents or acetylcholinesterase inhibitors induce exaggerated effects on REM density and latency in depressed subjects than in healthy controls (Berger et al., 1983;Gillin et al., 1991a;Nofzinger et al., 1997;Nurnberger et al., 1989). In addition, both manic and depressed bipolar subjects show increased pupillary sensitivity to the muscarinic cholinergic agonist pilocarpine relative to controls (Sokolski and Demet, 1996). Despite the data implicating the mAChR receptor system in mood disorders, no direct in vivo investigations of the central mAChR have been performed in depressed subjects. A novel PET radioligand, 18FFP-TZTP was recently developed by Eckelman (2001a;b) as a selective agonist of M2 receptors. Because the M2 receptor functions predominantly as a presynaptic release-controlling autoreceptor, decreased distribution volume (V) of this receptor could conceivably give rise to increased postsynaptic muscarinic receptor sensitivity. This project conducted a PET study of M2 receptor distribution volume in currently depressed subjects with major depressive disorder, currently depressed subjects with bipolar disorder, and psychiatrically healthy controls, about 25 subjects being studied in the past one year. The results confirmed the central hypothesis that M2 receptor V is decreased in regions where they are primarily located presynaptically in depressed subjects with bipolar disorder relative to healthy controls, namely in the cingulate cortex, insula, corpus callosum, and ventral striatum. These regions have been implicated in other studies as areas where impaired cholinergic regulation may result in abnormal emotional and attentional processing and altered emotional experiences including dysphoria, anxiety and euphoria. As such these data advance knowledge regarding the pathophysiology of depression. The magnitude of the cholinergic receptor abnormality correlated with the emotional salience which these subjects attributed to positively and negatively valenced words. Two scientific manuscripts have been prepared and submitted to describe these findings. During this past year, we demonstrated that this abnormality in bipolar disorder is specifically accounted for by subjects who are homozygous for a mutation of the M2 receptor. We are preparing a manuscript describing this seminal finding. We are now attempting to replicate this finding of the relationship between M2 receptor binding and M2 receptor polymorphism genotype. Moreover, we noted that the bipolar subjects who had this genotype and the associated reduction in M2 receptor binding manifested a more disabling and severe form of bipolar depression. We will thus be characterizing the bipolar disorder subgroup that exhibits this abnormality and genotype to identify clinical and biological correlates of this group. If these findings can be replicated in our expanded sample, we may be able to establish a particularly severe, genetically identified subtype of bipolar disorder. This paper has now under reviewed for publication. Finally, the genetic sampling from the subjects in this neuroimaging study were used to identify single nucleotide polymorphisms (SNPs) associated with unipolar depression (MDD) in a sample enriched for the likelihood of having genetic liability for MDD based upon having recurrent illness and early age-at illness-onset. The associations discovered in this very-well characterized, enriched MDD sample were then replicated in a larger sample selected more generally according to MDD criteria alone. One genetic marker showed a significant dominance genetic association effect with the MDD diagnosis which also remained significatnt (p<0.004) after correcting for multiple testing. Replication testing in a second, larger sample of healthy controls (n=739) and MDD cases (n=1,915) derived from the STAR*D cohort, confirmed the association with this marker(p<0.02). The marker so implicated mapped to the HTR3C gene, which encodes expression of the serotonin type 3C receptor subunit. The SNP variation (A/G) of this marker is located in the 3 untranslated region, which has a cis/trans control region known to subserve regulatory mechanisms such as translation and transport. Our results suggest that variation in HTR3C is a risk factor for MDD. A paper describing this finding is in the final stages of preparation.